In an effort to understand the physical and pharmacologic properties of benzodiazepine and barbiturate anticonvulsants, we will investigate GABA/benzodiazepine/-barbiturate ligand and receptor interactions. We will characterize GABA evoked single channel chloride currents and the regulation of these currents by benzodiazepine and barbiturate receptor agonists and inverse agonists. The anticonvulsant activity of benzodiazepines and barbiturates may in part be mediated by potentiating GABA inhibition of neuronal excitability. Intracellular recordings have shown the potentiation to be produced by increasing and/or prolonging GABA-evoked whole cell chloride currents but the mechanisms appear to be different and are poorly understood. The whole cell chloride currents are composed of many smaller ionic currents conducted through channels in the membrane and the channels open and close briefly when activated. A key to understanding how the anticonvulsants effect their differential response is to investigate the coupling between ligand binding and the gating properties or kinetic behavior of opening and closing of the chloride channel. Ligands may alter the frequency of channel opening and closing through modification of receptor binding and/or alter the physical properties of channel gating. Intracellular recording techniques and noise frequency analysis cannot resolve the complex behavior and mechanisms of channel gating. Newly developed patch clamp techniques, which enable direct recording of single ion channel currents, will be used in performing studies on mouse spinal cord and cortical neurons in primary dissociated cell culture. Emphasis will be placed upon identification and characterization of the conductances involved and the mechanisms of action between ligand binding and the regulation of opening and closing of the GABA/benzodiazepine/barbiturate chloride channel complex. The goal is to establish how benzodiazepine and barbiturate anticonvulsants potentiate GABA inhibition by investigation of their actions ar the single ion channel/ receptor level.
